A highly selective fluorescence method with Amplex Red HRP was developed to determine the hydrogen peroxide in mainstream cigarette smoke. This method’s LOD=13.5 nmol/L, recovery 98.3 %-101.7 % and RSD<3.17 % (n=5). The level of hydrogen peroxide in mainstream smoke was of micrograms per cigarette and positively correlated with tar delivery. For the cigarettes with the same tar delivery, the delivery of hydrogen peroxide was dependent on their design parameters. This method is suitable for determining the delivery of H2O2 in mainstream cigarette smoke.